Deoxyribonucleic acid (DNA) is the genetic information carrier found predominantly in the nucleus of living cells and constitutes the primary genetic material of all cellular organisms and of DNA viruses.
DNA is a linear or circular double-stranded helical polymer, each strand containing a sugar-phosphate backbone composed of deoxyribose sugar moieties substituted by phosphate groups at their 5′ and 3′ hydroxyls, the sugar groups being connected to 4 purine and pyrimidine bases usually indicated by their initial letters, namely, adenine (A), guanine (G), cytosine (C) and thymine (T). The two single strands of the DNA (ssDNA) are connected through hydrogen bonds between the complementary base pairs A-T and C-G (the Watson-Crick base pairs), of the two strands.
Genes are segments of DNA molecules containing all the information required for synthesis of a product, namely, a protein/polypeptide chain with a certain biological function or an RNA molecule. A mutation in a gene, e.g., a change in one or more base pairs of the normal gene, may result in a protein product with a change in biological function and, thus, in a genetic defeat or disease.
In the biological process of synthesis of polypeptide chains, the first step is transcription whereby the double-stranded DNA (dsDNA) serves as a template for synthesis of a single-stranded ribonucleic acid (RNA) with a base sequence complementary to one strand of the double-stranded DNA. In the second step, the translation, the polypeptide chain is synthesized using the RNA as a template. The amino acid sequence of the protein is completely determined by the sequence of bases in the RNA, which in turn is determined by the sequence of bases in the DNA of the gene from which it was transcribed.
The two single strands constituting a double-stranded DNA (dsDNA) can be separated by heating the DNA to ˜90° C. or by increasing the pH to extreme values, in a process called denaturation. The denatured complementary strands can react again to form the dsDNA by slow cooling of the DNA to the body temperature or by reducing the pH back to neutral, in a process called hybridization.
The hybridization process provides a powerful technique to detect mutations in a DNA sequence due to the selective binding of a single-stranded DNA (ssDNA) to its complementary strand. This specific property of the DNA is referred to as the “recognition” process.
The selectivity of the hybridization is affected by the conditions in which this process is done, i.e., the solvent (ionic strength) and the temperature. It is expected that the hybridization kinetics will depend on the number of complementary bases. The difference in hybridization rates, when the number of complementary bases changes, is an indication of the selectivity of the process.
Fast and efficient determination of DNA sequences and, particularly, the identification of mutations occurring in DNA sequences, is of major importance in the diagnosis of genetic diseases and in certain cases of cancer.
Several methods have been developed in recent years for the detection and isolation of specific DNA sequences, for example for diagnostic applications. Some of these methods are based on molecular hybridization whereby formation of a partially or fully complementary nucleic acid duplex occurs by association of single strands, usually between DNA and RNA strands, but also between RNA strands. In the technique of in situ hybridization, a known nucleic acid sequence, single stranded and usually with radioactive or fluorescent labels, is applied to a DNA-containing sample and annealing occurs in situ.
Conventional techniques exist for automated DNA sequencing and mutation detection. However, some of these methods include radioactive labeling, which require special handling techniques. In addition, obtaining results using the old methods may take several days. The computerized methods with similar principles are faster but expensive.
The sensors that are currently used for mutation detection are usually based on a probe ssDNA that is immobilized on a substrate, which can be silicon, polymer, gold, etc. The substrate is connected to a transducer that translates the signal of the events occurring on the surface into a physically measurable parameter. The addition of the target DNA and its level of hybridization to different probes on the substrate is indicated by a change in the signal. The hybridization between two fully complementary strands yields a change in the signal, that should be distinguished significantly from the hybridization between two non-complementary or partially complementary strands.
The currently developed detectors can be categorized according to the detection methods they use. Electrochemical-, fluorescent- and transistor-based detectors are known wherein the first step in their development is the immobilization process, in which a DNA strand is connected to a substrate.
One example of DNA sensors, based on fluorescence detection, is described by Nemoto et al. (U.S. Pat. No. 5,556,529) or by Livache et al., 1994. In that method, DNA sequences are amplified by replication for better sensitivity and then transcribed to double-stranded RNA. The synthesis of double-stranded RNA is quantified by fluorescence.
A second example of DNA sensors is electrochemically-based (Wang et al., 1996), in which a single-stranded DNA, which is adsorbed on an electrode, reacts with a complementary strand and the reaction is monitored by chronopotentiometric measurements.
Another detection method of biological reactions is based on the use of a field effect transistor (Schenk, U.S. Pat. No. 4,238,757) whereby the current between two electrodes in a silicon-based transistor is measured as a function of the voltage on a third electrode inserted in a biological solution, which is placed in the gap between the two electrodes.
PCT Publication No. WO 98/19151 (Cahen et al., 1998), of the same applicants of the present application, herein incorporated by reference as if herein described in its entirety, describes a hybrid organic-inorganic semiconductor device and sensors based thereon, said device characterized by being composed of:
(i) at least one layer of a conducting semiconductor;
(ii) at least one insulating layer;
(iii) a multifunctional organic sensing molecule directly chemisorbed on one of its surfaces, said multifunctional organic sensing molecule having at least one functional group that binds to said surface and at least one other functional group that serves as a sensor; and
(iv) two conducting pads on the top layer making electrical contact with the electrically conducting layer (i), such that electrical current can flow between them at a finite distance from the surface of the device.
These molecular controlled semiconductor resistors, herein designated MOCSER, are hybrid organic-inorganic semiconductor devices composed of one or more insulating or semi-insulating layers, one conducting semiconductor layer, two conducting pads, and a layer of multifunctional organic molecules, characterized in that: (i) said conducting semiconductor layer is on top of one of said insulating or semi-insulating layers; (ii) said two conducting pads are on both sides on top of an upper layer which is either said conducting semiconductor layer or another of said insulating or semi-insulating layers, making electrical contact with said conducting semiconductor layer; and (iii) said layer of multifunctional organic molecules is directly bound through at least one of said functional groups to the surface of said upper layer, between the two conducting pads, and at least another of said functional groups of said multifunctional organic molecules binds chemicals or absorbs light. The multifunctional organic molecules are directly bound to the surface of said upper conducting semiconductor layer or insulating or semi-insulating layer through at least one functional group selected from one or more aliphatic or aromatic carboxyl, thiol, acyclic sulfide, cyclic disulfide, hydroxamic acid and trichlorosilane groups. The at least another functional group of said multifunctional organic molecules that binds chemicals may be a metal-binding and metal-detecting group selected from radicals derived from hydroxamic acids, bipyridyl, imidazol and hydroxyquinoline that can detect a metal ion such as Cu2+, Fe2+ and Ru2+ metal ions. The at least another functional group of said multifunctional organic molecules that absorbs light is selected from aliphatic or aromatic hydroxamates, substituted aromatic groups such as cyanobenzoyl and methoxybenzoyl, bipyridyl groups, hydroxyquinoline groups, or imidazolyl groups to which a metal porphyrin or a metalophthalocyanin residue is attached. Examples of said multifunctional organic molecules are 2,3-di(p-cyanobenzoyl) tartaric acid (DCDC), 4,5-di(p-cyano-benzoyloxy)-1,2-dithiane (DCDS), 4,5-di(p-methoxy-benzoyloxy)-1,2-dithiane (DMDS) and 1,2-dithiane-4,5-di(hydroxyquinoline) and the Cu2+ complex thereof.
The device described in WO 98/19151 serves as a generic amplifier for chemical processes occurring on its surface. In one example, the device is based on a GaAs/(Al,Ga)As structure which is built in such a way that the current through it is extremely sensitive to the electric potential on its surface. As a result the device is very sensitive to any change in the charge distribution in molecules that are adsorbed on its surface (Gartsman et al., 1998; Vilan et al., 1998).